Fig 2: The DROSHA and AIM2 expression levels were elevated in alveolar macrophages of patients with IPF. (A) Representative immunofluorescence image of CD68 (Green), DROSHA (Red), and DAPI (Blue) staining in lung tissues from patients from IPF (IPF) or healthy subjects (Control). Positive area and cells are indicated by white arrows. Scale bars, 200 μm. Quantification of co-localization positive cells between DROSHA and CD68 (The percent of co-localization positive cells in total 100 cells in 10 individual images per group) (right) in lung tissues from patients with IPF (IPF, n = 5) or non-IPF patients (Control, n = 5). (B) Representative immunofluorescence image of CD68 (Green), AIM2 (Red) and DAPI (Blue) staining in lung tissues from patients with IPF or non-IPF patients (Control). Positive area and cells are indicated by white arrows. Scale bars, 200 μm. Quantification of co-localization positive cells between AIM2 and CD68 (the percent of co-localization positive cells in total 100 cells in 10 individual images per group) (right) in lung tissues from patients with IPF (IPF, n = 5) or non-IPF patients (Control, n = 5). (C) Representative immunofluorescence image of AIM2 (Green), DROSHA (Red), and DAPI (Blue) staining in lung tissues from patients with IPF or non-IPF patients (Control). Positive area and cells are indicated by white arrows. Scale bars, 200 μm. Quantification of co-localization positive cells between DROSHA and AIM2 (the percent of co-localization positive cells in total 100 cells in 10 individual images per group) (right) in lung tissues from patients with IPF (IPF, n = 3) or non-IPF patients (Control, n = 3). Data are mean ± SEM. *** p <0.001, ** p <0.01; by Student’s two-tailed t-test.

Fig 3: Drosha is phosphorylated at multiple sites in its RS-rich domain by Cdk5.a WB following immunoprecipitation showing the phosphorylation of Drosha and the Drosha truncation mutant Drosha-∆220-FLAG in HEK-293T cells expressing both Cdk5 and p25. b WB following immunoprecipitation showing the phosphorylation of Drosha and the Drosha truncation mutant Drosha-∆390-FLAG in HEK-293T cells expressing both Cdk5 and p25. c In vitro kinase assay showing the phosphorylation of purified Drosha-wt-FLAG, Drosha-∆220-FLAG and Drosha-∆390-FLAG. d WB following immunoprecipitation showing the phosphorylation of single-amino acid Drosha mutants in HEK-293T cells expressing both Cdk5 and p25. e WB following immunoprecipitation showing the phosphorylation of the multiple-amino acid Drosha mutant Drosha-mt5-FLAG in HEK-293T cells expressing both Cdk5 and p25. f Quantification of the protein levels of Drosha and Drosha mutants in HEK-293T cells expressing both Cdk5 and p25 or not by WB (n = 4, **P < 0.01). The data are presented as the means ± SEMs.

Fig 4: Deficiency of DROSHA suppresses AIM2 inflammasome activation in macrophages. (A) Quantification of IL-1β (left), IL-18 (middle) and TNF-α (right) secretion from WT bone marrow-derived macrophages (BMDMs) transduced with DROSHA -targeting small interfering RNA (Drosha siRNA), or with a control plasmid (Control), and stimulated with LPS and poly(dA:dT) (n = 9 mice per group). (B) Representative immunoblot analysis for caspase-1 and IL-1β (left) and densitometry quantification of caspase-1 p10 and IL-1β p17 levels (normalized to levels of β-actin) (right) from WT BMDMs transduced with DROSHA -targeting siRNA (Drosha siRNA), or with a control plasmid (Control), and stimulated with LPS and poly(dA:dT). For immunoblots, β-actin was used as loading control (n = 3 mice per group). (C) Quantification of IL-1β and IL-18 secretion from WT BMDMs transduced with DROSHA -targeting siRNA (Drosha siRNA), or with a control plasmid (Control), and stimulated with LPS and either ATP, flagellin, or MDP (n = 9 mice per group). (D) Representative immunofluorescence images (total 100 cells in 10 individual images per group) (left) and quantification (right) of ASC speck formation (white arrows) (the number of ASC speck positive cells in 10 individual images per group) in WT BMDMs transduced with DROSHA -targeting siRNA (Drosha siRNA), or with a control plasmid (Control), and stimulated with LPS and poly(dA:dT). (n = 6 mice per group). Scale bars, 20 μm. Data are mean ± SEM. ** p <0.01; by Student’s two-tailed t-test or ANOVA.

Fig 5: The DROSHA protein levels were elevated in alveolar macrophages during bleomycin-induced pulmonary fibrosis. (A) Representative immunohistochemistry image of DROSHA in lung tissues from wild-type (WT) mice were exposed to PBS or bleomycin via oropharyngeal aspiration. Positive area and cells are indicated by the black arrow. Scale bars, 200 μm. Results are representative of three independent experiments. (B) Representative immunoblot analysis for DROSHA and DGCR8 (left) and densitometry quantification of DROSHA and DGCR8 levels (normalized to levels of β-actin) (right) in lung tissues from WT mice exposed to PBS (n = 3) or bleomycin (n = 5) via oropharyngeal aspiration. For immunoblots, β-actin was used as loading control. Data are representative of three independent experiments. Data are mean ± SEM. * p <0.05; by Student’s two-tailed t-test. (C) Representative immunofluorescence image of F4/80 (Red), DROSHA (Green), and DAPI (Blue) staining in lung tissues from WT mice exposed to PBS or bleomycin via oropharyngeal aspiration. Positive area and cells are indicated by white arrows. Scale bars, 200 μm. Quantification of co-localization positive cells between DROSHA and F4/80 (the percent of co-localization positive cells in total 100 cells in 10 individual images per group) (right) in lung tissues from WT mice exposed to bleomycin (n = 5) or PBS (n = 3) via oropharyngeal aspiration. Data are mean ± SEM. ** p <0.01, * p <0.05; by Student’s two-tailed t-test.